The 20,000-Da subunits of vertebrate nonmuscle myosins are phosphorylated by specific calcium-calmodulin dependent kinases, myosin light chain kinases. This phosphorylation regulates the actin-activated ATPase activities of these myosins and appears to play a central role in the regulation of cellular motile activities. However, there is disagreement as to the mechanism of this regulation. Other investigators have reported that phosphorylation causes large increases in the actin-activated ATPase activities of these myosins and have hypothesized that this phosphorylation acts as an on/off switch. However, we have found that at high actin concentrations or in the presence of tropomyosin, the ATPase activity of filamentous cell thymus myosin is almost independent of phosphorylation. In an attempt to reconcile the difference between our results and those of other investigators, we have determined the actin-activated ATPase activity and filament stability of thymus myosin under a variety of conditions. Both the unphosphorylated and phosphorylated myosins needed to be filamentous for their ATPase activities to be activated by actin. Under conditions where phosphorylation increased myosin filament formation, there were large increases in actin-activated ATPase activity upon phosphorylation, but under conditions where phosphorylation had only a small effect on myosin filament formation it had only a small effect on actin-activated ATPase activity. These results suggest that phosphorylation may regulate actomyosin based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly. As cytoplasmic myosins are also phosphorylated in vivo by the calcium-phospholipid dependent protein kinase, protein kinase C, we have examined the effect of protein kinase C phosphorylate on the actin-activated ATPase activity and filament assembly of thymus myosin.